Myocardial ischemia/reperfusion (I/R) injury is the most common cause of death in the US. In experimental models, relatively young animals can be protected from I/R injury through activation of protective signaling pathways, but this protective effect is much more difficult to induce in the presence of co-morbidities. Since coronary artery disease occurs primarily in patients with co-morbidities, experimental cardioprotective interventions may not be applicable to the most relevant human population. In addition, males and females have different sensitivities to I/R injury. One signaling pathway that has major importance in cardioprotection is the activation of nitric oxide synthase, which can result in nitrosylation of protein cysteine residues. Directly inducing cysteine modification may be a way of bypassing defective signaling pathways and directly affecting downstream targets of cardioprotection. In this proposal we will study two post-translational modifications (PTMs) of cysteine, S-nitrosylation (SNO) and S-sulfhydration (SSH), both of which have cardioprotective characteristics, and which may interact. Progress in this area has been hampered by the lack of sensitive methods for detecting specific cysteine modifications, and relating this to cardioprotection. One of the benefits of SNO, and potentially SSH, is that it is reversible and it may block cysteines from other oxidative modifications during I/R, preventing irreversible oxidation. To assess the importance of SNO and SSH, we have developed and are developing methods that can detect PTMs of specific cysteine residues in specific proteins. This proposal is to employ these methods that allow SNO, SSH, and oxidation of specific cysteine residues to be measured using several mass spectrometry approaches, and allow the proportion of cysteines that are modified to be quantified. Aim 1 will examine the molecular mechanisms regulating SNO. We will test the hypothesis that transnitrosylation confers specificity to mitochondrial SNO, regulating which proteins are S- nitrosylated. We will assess the mechanisms that localize SNO signaling and examine how caveolar signaling by eNOS leads to preferential SNO of subsarcolemmal mitochondria. Aim 2 will test the hypothesis that SSH enhances cardioprotection in part by cross talk with SNO. We will examine the role of protein SSH in cardioprotection, determining specific sites of SSH, sex differences in SSH, and the interaction between SSH and SNO modifications. Aim 3 will evaluate the effect of SNO and SSH on the function of key proteins by gene editing the SNO sites using novel TALEN and CRISPR methods to generate mice in which key cysteines are mutated to serine or alanine to evaluate the effect of these PTMs on cardioprotection. The innovative aspects of the project are the novel hypotheses, the novel methods for directly identifying and quantifying SSH, and the development of novel genetic mouse models to test the role of specific PTM sites in cardioprotection. The results of these studies will provide insights into the mechanisms of cardioprotection and may suggest approaches to bypass defective signaling pathways to directly protect the heart from I/R injury.